These enzymes are typically conjugated to secondary antibodies, which bind to the primary antibody against the protein of interest. Primary antibodies directly conjugated to the enzyme can also be used. The most commonly used enzymes are horseradish peroxidase (HRP), which converts 3,3'-diaminobenzidine (DAB) into a brown product, and alkaline phosphatase (AP), which converts 3-amino-9-ethylcarbazole (AEC) into a red product.

Due to the higher signal amplification involved in this method, chromogenic detection is usually more sensitive than fluorescent detection. Furthermore, unlike fluorophores, the colored precipitates created from substrates such as DAB are photostable, enabling storage of the slides for many years. Unlike fluorescent detection, which requires specialized light sources and filters, chromogenic detection only requires a standard microscope. However, the experimental procedure is longer as it includes more incubation and blocking steps than fluorescent methods.

Four main methods of indirect chromogenic detection are widely used today. The biotin-based methods use an avidin-biotin complex (ABC) or a labeled streptavidin-biotin binding (LSAB) complex. The non-biotin based methods employ a polymer complex or a micropolymer complex.

Avidin-biotin complex (ABC) method

Early use of the ABC method relied on biotinylated secondary antibodies and an avidin-biotin- reporter enzyme complex. As avidin is tetravalent, large complexes form, resulting in high signal intensity.

Four main methods of indirect chromogenic detection are widely used today. The biotin-based methods use an avidin-biotin complex (ABC) or a labeled streptavidin-biotin binding (LSAB) complex. The non-biotin based methods employ a polymer complex or a micro-polymer complex

Labeled streptavidin-biotin (LSAB) method

Most detection now relies on the LSAB variant of the ABC method, which uses streptavidin, instead of avidin. This results in less non-specific tissue binding, as streptavidin is not glycosylated and has a more neutral isoelectric point than avidin.

Polymer methods

The key challenge of biotin-based systems is that endogenous biotin can lead to significant background staining in certain tissues (e.g., brain). Whilst formalin fixation and paraffin embedding reduce biotin levels, antigen retrieval can result in biotin being exposed. In frozen sections, endogenous biotin is a significant problem. Although extra steps with biotin blocking solutions can be used to reduce background, non-biotin polymer-based methods offer an alternative. Early polymer methods used a dextran backbone to which multiple enzyme molecules and secondary antibodies are attached.

Superior micro-polymer / compact polymer methods use a smaller detection complex with less tendency to aggregate. This results in greater sensitivity through better tissue penetration and reduced background staining from endogenous biotin.

Multicolour detection IHC

If multiple antigens are of interest then it is possible to stain up to three different antigens at the same time using different colored chromogens. This typically requires primary antibodies that are raised in different species, unless the antigen is present at a high enough level that a primary antibody directly conjugated to the reporter enzyme can be used.

Blocking agents can also be used that enable staining with one primary antibody raised in a particular species, followed by blocking of secondary antibody binding sites on that primary antibody, and then staining with a second primary antibody raised in the same species.